The cytosolic signal transduction pathways from the receptor to the nucleus follow the interactions of extracellular growth factors with their receptors embedded in the cell membrane and involve an intracellular complex chain of biochemical events, still partially unknown, that leads signals to the nucleus in order to stimulate the proper biological response. In the signal transduction eliciting mitogenesis, an activated receptor transmits in some way a signal to cytosolic or membrane anchored proteins like one appearing to have a pivotal role in the mechanism, namely the p21.sup.ras protein. In turn these proteins are able to transduce further the signal in a multistep way to the nucleus. There are several human tumors in which activated ras proto-oncogenes have been detected. The frequency of activation may be very high; for example, activated ras gene occur in 30% of all human haemopoietic neoplasms and more than 90% of tumors of the exocrine pancreas. In general protooncogenes lead to normal proteins, like tyrosine kinase receptors and transducers, heavily involved in the cell signalling. Several oncogenes are mutated or overexpressed forms or chimeric genes from chromosomal translocation, coding for proteins that act in the signal transduction mechanism, and giving rise to constitutively activated signalling proteins that cause an uncontrolled cell growth resulting in tumor progression. Intracellular signalling is being elucidated more and more in the nature of its components and in their role, disclosing a more and more attractive set of possible targets for conceptually new approaches to antitumoral therapy. The signalling from the activated receptor to the nucleus seems to involve several proteins interacting along maybe redundant pathways, more or less interconnected. Among the main types of molecular events involved, some are of particular relevance: 1) protein tyrosine phosphorylation (kinase activity); 2) recognition (physical association between specific protein domains); 3) protein serine/threonine phosphorylation (kinase activity); 4) dephosphorylation by specific enzymes (phosphatases), because both tyrosine and serine/threonine phosphorylation are also regulated through the reversing effect. These events can occur more than once in the net of the signal transduction pathways. Among these main interactions, an appealing target for a therapeutical approach is the recognition of the phosphorylated tyrosine domain with the "cytosolic transducers".
As first step the growth factor (for example EGF, FGF, PDGF, HGF, . . . ) activates its receptor, a protein spanning the cell membrane, by interacting with it and causing mutual proximity of the receptor molecules. Indeed the receptor molecules are brought close to each other allowing inside the cell a reciprocal phosphorylation at the level of intracellular domains operated by the kinase domain of one molecule on several tyrosine residues of the cytosolic portion of the adjacent molecule, or undergo a phosphorylation by an associated tyrosine kinase. Once the cytosolic regions of the receptor have several sites phosphorylated at tyrosine a molecular recognition can occur between these phosphorylated sites and proper domains of several other proteins.
These proteins are "cytosolic transducers"; indeed they provide to continue the pathway of the signal transduction linking the ligand binding to the receptor to the regulation of nuclear factors controlling the transcription of genes and hence ultimately to the generation of biological responses such as cell proliferation (J. Schlessinger and A. Ullrich, Neuron, 1992, 9, 383; J. Schlessinger, Trends Biochem. Sci., 1993, 18, 273.). They all have a domain named SH2 (Src Homology 2) with a high degree of homology in the primary structure and even more conserved in the tertiary structure. These short (approximately 100 amino acids) SH2 domains are non-catalytic regions that are responsible for the binding with phosphotyrosine containing proteic segments (T. Pawson and G. D. Gish, Cell, 1992, 71, 359.). The interest and studies on structure and role of these SH2 domains have raised sharply in the last years. An analogous interest has been elicited by SH3 domains (Src Homology 3), less caracterized in function but often accompaning the presence of SH2 domains in the cytosolic transducers. SH3 domains also are non catalytic regions with high homology and that seem to have affinity for glycine-proline rich domains. Both SH2 and SH3 regions are thought to have a pivotal role in the signal transduction for intramolecular recognition. One group of SH2 containing proteins includes proteins having an intrinsic enzymatic activity, hence endowed with catalytic domains (kinase domains or others) besides the SH2 domain, like the proteins Src, Abl, Syc, PTP1C, PLCg, GAP, vav. Another group comprises SH2 containing proteins devoid of any known catalytic domain, that seem to have the function of adaptors recruiting to the receptor other proteins endowed with catalytic properties. For example this is the case of p85 that provides a link between the activated (phosphorylated) receptor and the activity of phosphatidyl-inositol-3'-kinase (PI3K). Another relevant example of adaptor is Grb2 (growth factor receptor binding protein 2) that links the phosphorylated receptor (EGF-R, PDGF-R, HGF-R, . . . ) to a protein that has a guanine nucleotide exchange activity on p21.sup.ras, activating a system like ras, directly related to mitogenesis. Other SH2-containing adaptors seem to be Shc, Crk, Nck. Some of the mentioned transducers, particularly Grb2, and the related binding site on receptors like HGFR, articularly the site comprising Tyr 1356 of HGF receptor, seem to be strongly related to cell motogenesis, to invasiveness, and finally to metastasis. The peculiar SH2 domain is able to recognize the phosphorylated tyrosines and the flanking sequences of the receptors. or some other intermediate proteins. The core element of the rather conserved structure is an antiparallel .beta.-sheet that is sandwiched between two .alpha.-helices and a small .beta.-sheet protruding out from the sandwich; this structure has been clarified by X-ray and NMR studies (G. Waksman, D. Kominos, S. C. Robertson, N. Pant, D. Baltimore, R. B. Birge, D. Cowburn, H. Hanafusa, B. J. Mayer, M. Overduin, M. D. Resh, C. B. Rios, L. Silverman and J. Kuriyan, Nature, 1992, 358, 646; G. Waksman, S. E. Shoelson, N. Pant, D. Cowburn and J. Kuriyan, Cell, 1993, 72, 779; M. Overduin, C. B. Rios, B. J. Mayer, D. Baltimore and D. Cowburn, Cell, 1992, 70, 697) also in the complex form with binding peptides, like in the case of the Src SH2 domain. Anyway loops and turns differ among the several transducers, and can be related to the recognition specificity. Analyzing the phosphorylated receptor interactions with the cytosolic transducers, we can see: 1) that each of the several receptors links with almost the same proteins and 2) that each of them can do it with a certain degree of regiospecificity, namely for several receptors different SH2 containing transducers bind different phosphotyrosine residues of the receptor cytosolic domain (J. A. Escobedo, D. R. Kaplan, W. M. Kavanaugh, C. W. Turck and L. T. Williams, Mol. Cell. Biol., 1991, 11, 1125; A. Kashishian, A. Kazlauskas and J. A. Cooper, EMBO J., 1992, 11, 1373; C.-H. Heldin, EMBO J., 1992, 11, 4251 ). The high specificity arises from the sequence surrounding the phosphotyrosine, particularly downstream, different recognition motifs occurring for each transducer (W. J. Fantl, J. A. Escobedo, G. A. Martin, C. W. Turck, M. del Rosario, F. McCormick and L. T. Williams, Cell, 1992, 69, 413 ). A phosphopeptide library has been done to analyze the sequence specificity of several cytosolic transducers and determining the relative consensus sequences (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen and L. C. Cantley, Cell, 1993, 72, 767).
Indeed for the receptor of Hepatocyte Growth Factor (HGF), that is a powerful mitogen for hepatocytes in primary cultures and the major mediator of liver regeneration in vivo (P. M. Comoglio, in Hepatocyte Growth Factor-Scatter Factor (HGF-SF) and the c-Met receptor. I. D. Goldberg and E. M. Rosen Eds. (Basel), Switzerland: Birkauser Verlag, 1993, 131-165 ), the interactions of all the cytosolic transducers seem to be concentrated on a same "supersite" involving two close phosphotyrosine residues (C. Ponzetto, A. Bardelli, Z. Zhen, P. dalla Zonca, F. Maina, S. Giordano, A. Graziani, G. Panayotou and P. M. Comoglio, Cell, 1994, 77, 261).
The growth factor receptor-bound protein 2 (Grb2) is a relatively small adapter simply constituted by a SH2 domain flanked by two SH3 domains and Grb2 forms a complex in vivo with another protein, SoS, through the binding of its SH3 domains with a 31 aminoacid proline-rich stretch located in the C-terminal domain of SoS , that in turn is able to interact with the ras system. Grb2, also in the complex form with SoS, is able to associate through its SH2 domain with specific tyrosine-phosphorylated sites of different receptors or transducers. Also the phosphorylated form of the cytosolic protein Shc can bind the SH2 domain of Grb2 (S. E. Egan, B. W. Giddings, M. W. Brooks, L. Buday, A. M. Sizeland and R. A. Weinberg, Nature, 1993, 363, 45 ). The connection of the Grb2/SoS complex with the Ras system is proved for HGFR (A. Graziani, D. Gramaglia, P. dalla Zonca and P. M. Comoglio, J. Biol. Chem., 1993, 268, 9165) and is largely supported by parallel cases with other receptors (EGFR , IRS-1) and in different biological species (J. Schlessinger and A. Ullrich, Neuron, 1992, 9, 383 ; J. Schlessinger, Trends Biochem. Sci., 1993, 18, 273 ; P. Polakis and F. McCormick, J. Biol. Chem., 1993, 268, 9157). The protein SoS is also known as "exchanger", Guanine nucleotide Dissociation Stimulator (GDS), and in its active form, complexed with Grb2 , is able to promote the fast GDP release from the inactive Ras-GDP complex (C. F. Albright, B. W. Giddings, J. Liu, M. Vito and R. A. Weinberg, EMBO J., 1993, 12, 339). Hence p.sub.21.sup.Ras can rapidly take GTP to form the active Ras-GTP, that in turn can act on the effectors of the Ras system transmitting the signal downstream to the nucleus (through the MAP kinase cascade). The membrane anchored complex Ras-GTP is the active form that acts on the effectors to transmit the mitogenesis signal to the nucleus. Its level is increased by the exchanger (GDS or SoS) activity and decreased by the GAP (GTPase activating protein) activity. Both these regulating enzymes are affected by growth factor receptor stimulation and furthermore their activity may depend from other regulatory factors. The two regulating enzymes, GDS and GAP, can have a different impact on Ras system in different cell type. Indeed, the mentioned regulations of the Ras system require a mature form of p.sub.21.sup.ras suitable to be anchored to the membrane (C. J. Marshall, Science, 1993, 259, 1865). In these cases cell culture results have confirmed that the Ras system is a valuable target for preventing relevant oncogenic transformations (J. B. Gibbs, A. Oliff, and N. E. Kohl, Cell, 1994, 77, 175). Continuing on the signal transduction pathway (downstream of Ras), the active form of the Ras system, namely Ras GTP, interacts with the N-terminus of its own effector, recently identified as the serine/threonine kinase Raf , thus initiating a cascade of phosphorylation by the "mitogen-activated protein kinases" (MAPs). Indeed, Raf provides the direct phosphorylation and hence activation of MAP kinase kinase (MEK) that in turn activates MAPK, leading to the activation of transcription (transcription factor AP1) in the nucleus (M. S. Marshall, Trends Biochem. Sci., 1993, 18, 250).